Bone marrow steady state erythropoiesis is primarily homeostatic, producing erythrocytes at a constant rate to replace the erythrocytes lost through normal turnover. However, during embryogenesis and in response to anemic stress, new erythrocytes must be produced at an accelerated rate. This process, termed stress erythropoiesis, utilizes a specialized population of stress progenitors that are phenotypically distinct from steady state erythroid progenitors. In preliminary data shown here we demonstrate, for the first time, an essential role for the scaffold proteins, Gab1 and Gab2, in stress erythropoiesis. Scaffold proteins form signaling hubs by integrating the information from a variety of external signals and transforming this information into the appropriate biological response. However, due to the high level of complexity of these scaffolds, the mechanisms by which Gab1 and Gab2 promote distinct biological functions have remained elusive. We demonstrate here that, while Gab1 and Gab2 are highly related, their functions in stress erythropoiesis are non-compensatory. Furthermore, we have developed a novel ex vivo model of stress erythropoiesis in which serially transplantable stress erythroid stem cells undergo specification and expansion, ultimately giving rise to stress BFU-E. Using this approach, we have shown that Gab1 plays an essential role in the differentiation of stress BFU-E from stress erythroid stem cells, as demonstrated by the inability of Gab-deficient cells to upregulate CD71 and Ter119. We also provide evidence that Gab2 plays an essential role in promoting the proliferation and/or survival of stress erythroid progenitors. The studies proposed here will determine the molecular mechanism by which these related scaffolds promote non-redundant functions in stress erythropoiesis. Toward that end, we propose three specific aims. In aim 1, we will test the hypothesis that Recruitment of Shp2 by Gab1 promotes sustained Erk activation at the cell membrane, thus promoting the differentiation of stress BFU-E from stress stem cells. In aim 2, we will test the hypothesis that Gab2 plays an essential role in the activation of Stat3 and the upregulation of Pu.1 expression, thus preventing premature differentiation and promoting survival of differentiating stress erythroid progenitors. Finally, in aim 3 we will test the hypothsis that Gab1 and Gab2 play essential roles in the ex vivo expansion of human stress erythroid progenitors. These studies will provide significant insight into the molecular mechanisms governing the rapid expansion of stress erythroid progenitor cells during times of acute need, and will be essential in the identification of new pharmacological targets as well as the development of novel cell-based therapies for the treatment of acute and chronic anemia. PUBLIC HEALTH RELEVANCE: Hereditary anemias are amongst the most common monogenic traits in man. These diseases, together with anemia from chronic disease or old age, as well as treatment-induced anemia as accompanies chemotherapy or radiation therapy, are significant public health problems. Stress erythropoiesis is the process of replacing lost red blood cells in response to acute need. We have identified novel signaling pathways, which play a central role in stress erythropoiesis, by promoting the differentiation and survival of a specialized population of stress erythroid progenitor cells. The studies proposed herein aim to identify the molecular mechanisms by which these functions are regulated. Understanding the signaling pathways that govern the expansion of stress progenitors will be a critical step in the development of cell-based therapies for combating chronic and acute anemia.